ABSTRACT The first steps in atherosclerosis are the transendothelial movement and subendothelial accumulation of lipoprotein lipid. Endothelial cell (EC) transcytosis of LDL involves two receptors, scavenger receptor-BI (SR-BI) and activin-like kinase 1 (ALK1). We showed that ECs also internalize undigested chylomicrons via SR-BI and process them in lysosomes, leading to storage of some lipid as lipid droplets and the release of small extracellular vesicles (sEVs) that cause lipid accumulation in macrophages. While many have considered chylomicrons as non-atherogenic and too large to cross the EC barrier, this concept is now outdated with the understanding that lipoprotein entry into the artery is a receptor-mediated process. The overall goal of this project is to determine how EC chylomicron uptake affects EC biology, delivers lipids to the artery, and accelerates atherosclerosis. In Aim 1, we propose to determine how triglyceride-rich lipoproteins affect lipid transfer across the vascular endothelium. We provide preliminary data suggesting that N-terminal apoB18 has separate ligand binding regions for ALK1 and SR-BI. We will determine whether lipoprotein size or apoB length determines exposure of these two different regions. We also will determine how sEVs from chylomicron-conditioned ECs cause lipid accumulation in macrophages and determine the differences between sEVs released from control and chylomicron-treated ECs. In Aim 2, we propose in vivo studies to determine the role of the EC-chylomicron uptake pathway in tissue lipid delivery and atherosclerosis. We provide preliminary data suggesting that chylomicrons that accumulate in lipoprotein lipase (LpL) deficient mice increase atherosclerosis. We will use genetically modified mice and knockdown strategies to assess the uptake of LDL and chylomicron lipids into arteries and will assess whether more extensive atherosclerosis in mice with combined deficiency of LpL and the LDL receptor is due to hyperchylomicronemia. Finally, we will determine whether AAV-mediated overexpression of apoB18 reduces EC uptake of apoB lipoproteins in vivo and alters atherosclerosis. Completion of the proposed studies promises to alter our view of the relationship of chylomicrons to vascular disease, define a novel pathway for arterial accumulation of atherogenic lipids, and illustrate a possible approach to prevent these lipids from entering the artery.